title: PCNA is ubiquitinated by RNF8.
abstract: The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: Sufang
authors.initials: S
authors.lastname: Zhang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chea
authors.forename: Xiao
authors.initials: X
authors.lastname: Meng
authors.forename: Yajing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ernest Y C
authors.initials: EY
authors.lastname: Lee
authors.forename: Marietta Y W T
authors.initials: MY
authors.lastname: Lee
mesh_terms: D045744:Cell Line, Tumor; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D025821:Polyubiquitin / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C486521:RNF8 protein, human; D025821:Polyubiquitin; D008769:Methylnitronitrosoguanidine; C097310:UBE2D3 protein, human; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases